Formulier voor het aanvragen van materiaal en/of gegevens ten behoeve van wetenschappelijk
onderzoek bij de SKION
Aanpassing 19-4-2011 & flowsheet paragraaf 6 dd 5-2012
Naam aanvrager
1. M Kool / waarvoor M Hamdi
2. AYN Schouten-van Meeteren
Functie
1. Molecular biologist, member SIOP PNET biology group,Human Genetics
department, AMC, Amsterdam
2. Pediatric oncologist, Pediatric Oncology department, AMC, Amsterdam
Instituut
Emma Kinderziekenhuis AMC, Amsterdam
Datum
30-11-2010
Titel onderzoek
Feasibility study: standardized molecular biology in medulloblastoma for
future risk group stratification
Collaboration
P Wesseling, neuropathologist, UMC St Radboud, Nijmegen & VUMC
Amsterdam; E Aronica, neuropathologist AMC, Amsterdam
V de Haas, head laboratory SKION / DCOG Dutch Childhood Oncology
Group, The Hague, M Hamdi molecular biologist AMC
1. Overview of the current field of research
About 25% of childhood cancers are brain tumors, of which medulloblastoma, WHO grade IV 1, is the
most common malignant subtype. The prognosis of medulloblastoma patients ranges from 50 - 80%
survival depending on different risk factors at diagnosis. Current risk groups in medulloblastoma are
determined by the established clinical criteria of age, volume of the tumor residu, metastatic disease
and anaplastic large cell histology 1, 2. In addition to these clinical characteristics biologic properties of
the tumor cells have proven to be major factors influencing prognosis. Strong biologic predictors are
beta-catenin nuclear positivity for the favourable subgroup and MYC family amplification for the
unfavourable subgroup 3 ,4, 5.
Medulloblastoma is the most common malignant brain tumor of childhood and accounts for ~10% of all
childhood cancer deaths. Its clinical course is difficult to predict and, despite major improvements in
overall survival rates, current therapies are associated with significant long-term side-effects. In recent
years, a series of molecular and histopathological biomarkers have been identified which may allow a
more accurate prediction of disease outcome (eg. β-catenin status and nodular/desmoplastic histology
as favorable-risk markers, MYC family amplification and large-cell or anaplastic histology as high-risk
markers), and offer the potential for the development of more individualised therapies based on
disease-risk.
The next wave of clinical trials being planned for medulloblastoma are proposed to involve the
stratification of patients using molecular and histological biomarkers, alongside clinical indices, to
select favorable, standard and high-risk treatment groups. The first trials within SIOP will commence
late 2011 or early 2012. It is anticipated that the selection of patients for treatment on this basis will
underpin two concurrent trials; PNET5, which will test whether treatment can be reduced for a
favorable-risk disease sub-group, with the aim of maintaining survival rates while reducing late-effects,
and PNET6, which will aim to improve survival rates in the standard-risk group. Also within the COG
medulloblastoma protocols will become stratified partially on biological risk factors.
Patients in the Netherlands (~20-25 annually) are treated across different seven centres with pediatric
neurosurgery the success of these trials will depend on the collection of tumor material from these
multiple centres across the Netherlands, followed by its standardized central molecular genetic
analysis and histopathological review, prior to the commencement of adjuvant therapy. This must take
place within a 21-day window following initial surgical resection of the tumor, to allow the start of
therapy (chemo- and radio-therapy) received from day 28 onwards, based on the results obtained.
However, such a sample collection, handling and analysis system is not currently in place in the
Netherlands and requires establishment. This study will determine the feasibility of adequate and
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timely collection, transportation and central molecular biological analysis of prognostic biomarkers,
alongside central pathological review, in samples of tumor material from the primary resection of
patients with medulloblastoma, treated in multiple centres across the Netherlands.
Successful completion of this study will lead to the incorporation of this review system into trial
protocols, and the therapeutic stratification of medulloblastoma patients using molecular biomarkers,
An example of such are the forthcoming pan-European SIOP Phase III clinical trials. Finally,
development of this review system will form a strong basis for the real-time collection, banking and
analysis of other paediatric brain tumor types, to facilitate ongoing clinical and research studies.
2. Aim of the study.
To determine the feasibility of collection, transportation, centralised histopathological review and
molecular biological analysis of prognostic biomarkers, in samples of tumor material from the primary
resection of patients with medulloblastoma, treated in multiple centres across the Netherlands, prior to
commencement of radiotherapy. The aim of the study is to determine possible pitfalls and limitations in
the procedure to obtain biology data in medulloblastoma in order to reach a complete result within 21
days. A time frame of one year inclusion time will be set as limit to detect any possibility for
improvement in obtaining consent, sample handling, logistics and administrative procedures until
definite result to decide on the treatment of the patient with medulloblastoma. Successful completion
of this study will lead to the therapeutic stratification of medulloblastoma patients using molecular and
histological biomarkers, as part of forthcoming pan-European SIOP Phase III clinical trials.
Specific aims:
1. To determine whether it is feasible to obtain molecular and histological factors to be able determine
medulloblastoma therapy in the future treatment protocols, within a 21-day period post-surgical
resection.
2. To establish whether the rapid submission of snap-frozen and formalin-fixed medulloblastoma
material from local centres to a central laboratory is practicable.
3. To establish and implement methodologies for tissue collection, shipping, handling and biological
testing.
4. To assess the quality of material received, and its suitability for successful biological testing.
5. To establish the key methodologies necessary for the biological assessment of relevant
medulloblastoma biomarkers, in both snap-frozen and formalin-fixed biopsy material.
6. To develop and instigate Quality Control (QC) measures and Standard Operating Procedures
(SOPs) to govern all of the above, in conjunction with our SIOP PNET group partners.
7. To describe improvements in obtaining consent, sample handling, laboratory procedures and
administrative issues based on the national and international experience from the feasibility study
for the upcoming medulloblastoma treatment protocols based on molecular biology profile.
3. Relevance of the study
Medulloblastoma is the most common malignant brain tumor of childhood and accounts for ~10% of all
childhood cancer deaths. Overall survival rates are now around ~60% but there is great variation in
survival depending on current clinical prognostic factors. For example high-risk cases (i.e. metastatic
disease, age <3 yrs at diagnosis) have a poor prognosis (<40% survival). In addition, there is marked
variability in outcome within current risk groups and the current stratification system, based on clinical
disease features alone, is imprecise. Moreover, current therapies are associated with significant longterm side-effects in the majority of patients. Improved treatment stratification using biological indices
thus offers the opportunity for improved patient management and disease outcome; the delivery of
intensified treatment for those with poor-risk disease, whilst reducing therapy for favourable-risk cases,
with the goal of maximising survival whilst minimising late effects.
In recent years, a series of molecular and histopathological biomarkers have been identified which
may allow a more accurate prediction of disease outcome, and offer the potential for the development
of more individualised therapies based on disease-risk. A range of molecular markers with prognostic
utility have now been identified in trial-based studies (Table 1). Nuclear imunoreactivity of β-catenin,
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indicative of Wnt/Wg pathway activation, has been consistently associated with a favourable outcome .
Amplification of the MYC oncogene has been associated with a poor prognosis in multiple studies,
while defects of chromosome 17 [5], and expression of the ERBB2 receptor tyrosine kinase [6], have
each been associated with an adverse outcome in isolated studies. Other defects, such as expression
of the MYC oncogene or the TRKC neurotrophin receptor, and amplification of the MYCN oncogene,
have shown prognostic significance in some studies. Among the histopathological variants of
medulloblastoma, an aggressive biological behaviour has been established for the overlapping largecell and anaplastic variants, which have been associated with a poor outcome in several clinical trial
cohort {McManamy}. In contrast, the medulloblastoma with extensive nodularity (MBEN), which is a
tumor of infancy, is associated with a good prognosis, and the wider desmoplastic variant (including
MBENs) has consistently been associated with a more favourable outcome in trials-based studies in
infants.
Table 1. Molecular and histopathological markers of disease-risk in medulloblastoma, showing
disease features which display consistent associations with prognosis in ≥2 clinical trials-based
studies. IHC, immunohistochemistry; FISH, fluorescence in situ hybridization; qPCR, quantitative
polymerase chain reaction; OS, overall survival; EFS, event-free survival; PFS, progression-free
m
u
survival; multivariate analysis; univariate analysis; NR, not reported; NS, not significant; COG,
Children’s Oncology Group. **Cases showing high-level gene amplification in >25% of tumour nuclei.
See text for references.
Towards therapeutic stratification using biological markers in medulloblastoma clinical trials
Whilst there is clearly overlap between high-risk clinical, pathological and molecular variables, a
schema for stratifying medulloblastomas on the basis of combinations of these variables, which are
predictive of high- and low-risk cases, are likely to allow a more targeted approach to the use of
adjuvant therapy in the future. Such stratification systems are currently being prospectively validated in
biological studies attached to the recently-completed SIOP PNET4 clinical trial (which will assess all
markers described above in conjunction with clinical features), to establish any applications in routine
diagnostic practice. The next wave of clinical trials being planned for medulloblastoma by the SIOP
and COG group are proposed to involve the stratification of patients using molecular and histological
biomarkers, alongside clinical indices, to select favorable, standard and high-risk treatment groups.
These trials will commence in late 2011 or early 2012. It is anticipated that the selection of patients for
treatment on this basis will firstly underpin two concurrent trials; PNET5, which will test whether
treatment can be reduced for a favourable-risk disease sub-group, with the aim of maintaining survival
rates while reducing late-effects, and PNET6, which will aim to improve survival rates in the standardrisk group remaining following removal of the favourable-risk cases. The treatment protocols for these
trials are currently being planned.
Challenges to therapeutic stratification in medulloblastoma
A series of significant logistical challenges exist to the routine assessment of biomarkers and
histopathological indices as a basis for therapeutic stratification in medulloblastoma. Because patients
with medulloblastoma in the Netherlands (~20-25 annually) are treated across several centres, the
success of such studies will depend on the instigation of standardised systems for the collection,
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storage and shipping of frozen and formalin-fixed tumour material from multiple centres across the
Netherlands, followed by its centralised molecular genetic analysis and histopathological review, prior
to the commencement of adjuvant therapy. This must take place within a 21-day window following
initial surgical resection of the tumor, to allow the stratification of therapy (chemo- and radio-therapy)
received from day 30 onwards, based on the results obtained. However, such a sample collection,
handling and analysis system, alongside the necessary quality control procedures, are not currently in
place in the Netherlands and require establishment. In order to inform the specialists involved in
medulloblastoma treatment information was given to neurosurgeons meeting in January 2010,
neuropathologists meeting May 2010 and pediatric oncologists meeting February and March 2010.
A pan-European strategy for medulloblastoma treatment stratification in forthcoming trials
A coordinated approach will be taken between SIOP PNET group partners across Europe, for the
development of standardised systems for real-time biomarker assessment and histopathological
review, to be used as part of forthcoming SIOP Phase III trials. One central coordinating centre has
been designated for each country, with the AMC in Amsterdam adopting this role for the Netherlands.
As a prelude to the commencement of the upcoming trials, a feasibility study will be undertaken in
each country.
Future treatment protocols for medulloblastoma will use biologic properties of the tumor to identify
patients for different risk categories such as favorable or standard risk group for medulloblastoma.
Those upcoming treatment protocols, for example within SIOP and COG are divided in different risk
categories according to clinical as well as biological criteria. The strategies for SIOP studies have the
following structure: PNET 5 will investigate whether treatment can be reduced for a favorable-risk
disease sub-group aiming at maintaining survival rates while reducing late-effects, while PNET 6 aims
at improving survival rates in the standard-risk group.
Planning of the PNET5 and PNET6 trials is ongoing in parallel with this feasibility study, with a view to
their commencement in late-2011 / early-2012. Once feasibility is demonstrated, submission of biopsy
material will be mandatory for individual patient entry into the PNET5/6 clinical trials in order to perform
adequate risk classification. Subsequently biological categories will probably be implemented as
standard in any future medulloblastoma protocol, investigational or in a prospective fashion to stratify
treatment groups 6, 7.
.
Finally, development of this standardized system will form a strong basis for the real-time collection,
banking and analysis of other paediatric brain tumor types, to facilitate ongoing clinical and research
studies across Europe.
4. Preliminary results
The Human Genetics laboratory at the AMC in Amsterdam has applied for reference lab for the
Netherlands within the SIOP PNET biology Group. The laboratory is well experienced in biology
studies in medulloblastoma and is coöperating with the neuropathologists to obtain timely
standardized beta-catenin results. Furthermore the investigators are members of the PNET Biology
Group of SIOP.
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5.
Short description of the methods of investigation
This will be a descriptive biological study which will be open to patients with newly diagnosed
medulloblastoma from all Dutch treatment centres, which have had a biopsy or tumor resection.
Patients will be treated as per current guidelines for the disease, and no changes in therapy will be
made on the basis of the results of the study.
This study is NOT part of a clinical trial. Only tumor material in excess of diagnostic requirements at
the local centre will be used. Successful completion of this study by carefully describing improvements
in the process based on one year of national experience will lead to the mandatory submission of
biological material for entry into the upcoming SIOP and COG medulloblastoma treatment trials with
the incorporation of this review system into the protocols.
Following surgical resection, snap-frozen and formalin-fixed tumor material excess to diagnostic
requirements will be collected and stored for all cerebellar tumors at local centres. Next the flow
diagram below will be followed for handling of material. Following a histopathologically-confirmed local
diagnosis of medulloblastoma, material from these tumors only will be shipped to the AMC in
Amsterdam under standardised conditions. This study will focus on establishing, and determining the
feasibility of using key methodologies that will be required for the routine analysis of medulloblastoma
biomarkers, in formalin-fixed (pathology review, β-catenin immunohistochemistry (IHC)) and snapfrozen material (MYC family fluorescence in situ hybridisation (FISH). SOPs for all procedures have
been developed in conjunction with our SIOP PNET group partners in other countries.
Central pathology review will be coordinated by Dr P. Wesseling (St Radboud Medical Center,
Nijmegen and Free Universitry Medical Center, Amsterdam), according to the 2007 WHO criteria [13].
β-catenin status will be assessed by IHC. Interphase FISH will be carried out using commercial FISH
probes to assess copy numbers of MYC and MYCN in relation to syntenic reference probes (8
centromere for MYC, LAF [2q11] for MYCN).
Remaining frozen tissue will be stored at the reference laboratory for use in subsequent approved
biological studies. The SIOP PNET biology group is an international collaboration aiming for joint
research. Currently future research questions are under development within this group on samples
collected within the participating European countries. Subsequent studies concerning this tumor
material can only be performed after approval by the DCOG.
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6.
Quantify material as needed = flowsheet
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Ethical considerations
This study has no consequences for the treatment of the patient with medulloblastoma, whose
material is encorporated, since it investigates feasibility of timely standardized and centralized
biological risk stratification only. This study as such requires no approval of a medical ethics
committee according to the Dutch law. This has been deliberated with the Ethics committee in the
AMC Amsterdam.
Since the tumor material will be send to the reference laboratory located outside the treatment hospital
including patient identification the fulfilment of an informed consent is mandatory. This will be
performed for each individual patient, preferably prior to surgery.
7.
List of literature in the field of research .
Reference List
1. Packer RJ, Vezina G. Management of and prognosis with medulloblastoma: therapy at a
crossroads. Arch.Neurol. 2008;65:1419-1424.
2. von Hoff K, Hartmann W, von Bueren AO et al. Large cell/anaplastic medulloblastoma: outcome
according to myc status, histopathological, and clinical risk factors. Pediatr.Blood Cancer 2010;54:369376.
3. Ellison DW, Kocak M, Dalton J et al. Definition of Disease-Risk Stratification Groups in Childhood
Medulloblastoma Using Combined Clinical, Pathologic, and Molecular Variables. J.Clin.Oncol. 2010
4. Pfister S, Remke M, Benner A et al. Outcome prediction in pediatric medulloblastoma based on
DNA copy-number aberrations of chromosomes 6q and 17q and the MYC and MYCN loci.
J.Clin.Oncol. 2009;27:1627-1636.
5. de Haas T, Hasselt N, Troost D et al. Molecular risk stratification of medulloblastoma patients based
on immunohistochemical analysis of MYC, LDHB, and CCNB1 expression. Clin.Cancer Res.
2008;14:4154-4160.
6. Ellison DW, Onilude OE, Lindsey JC et al. beta-Catenin status predicts a favorable outcome in
childhood medulloblastoma: the United Kingdom Children's Cancer Study Group Brain Tumour
Committee. J.Clin.Oncol. 2005;23:7951-7957.
7. Ellison DW, Onilude OE, Lindsey JC et al. beta-Catenin status predicts a favorable outcome in
childhood medulloblastoma: the United Kingdom Children's Cancer Study Group Brain Tumour
Committee. J.Clin.Oncol. 2005;23:7951-7957.
8. Pizer BL, Clifford SC. The potential impact of tumour biology on improved clinical practice for
medulloblastoma: progress towards biologically driven clinical trials. Br.J.Neurosurg. 2009;23:364-375.
9. de HT, Hasselt N, Troost D et al. Molecular risk stratification of medulloblastoma patients based on
immunohistochemical analysis of MYC, LDHB, and CCNB1 expression. Clin.Cancer Res.
2008;14:4154-4160.
10. Kool M, Koster J, Bunt J et al. Integrated genomics identifies five medulloblastoma subtypes with
distinct genetic profiles, pathway signatures and clinicopathological features. PLoS.ONE.
2008;3:e3088.
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8.
Curriculum vitae of applicant
M Kool studied Molecular Sciences at the Agricultural University in Wageningen where he graduated
in 1989. In 1994 he obtained his PhD degree in Virology (studied DNA replication iin baculoviruses) at
this same university. After a 5 year postdoctoral period at the Netherlands Cancer Institute in
Amsterdam where he studied multidrug resistance in cancer cells he started in 1999 his own research
group at the AMC studying the biology and genetics of childhood tumors medulloblastoma and
rhabdomyosarcoma. Since July 2004 his group is located at the Human genetics department of the
AMC. M. Kool is the Dutch representative in the European SIOP PNET Biology group.
AYN Schouten studied Medicine at the Vrije Universiteit UMC in Amsterdam where she graduated in
1986 cum laude and in 1989 she completed the pediatric residence program at the University of
Leiden. After a specialist training in pediatric hemato-oncology at the Vrije Universiteit UMC she
specialised in the clinical practice of pediatric neuro-oncology and retinoblastoma. Research on drug
resistance in the laboratory op pediatric oncology led to the thesis :"In vitro drug resistance in
retinoblastoma and childhood central primitive neuro-ectodermal tumors" which in april 2003 finalized
with a doctorate. Since November 2005 she is working at the Emma Children's Hospital AMC in
Amsterdam, with specific attention for childhood brain tumors and in collaboration with the Lab of
Antropogenetics for translational research regarding medulloblastoma.
At a national level she is chairmen of the brain tumor subgroup of SKION (DCOG) and the protocol
committee for Low Grade Glioma. International of the Brain Tumor Committee of SIOP (Societé
Internationale Oncologie Pediatrique) and the subcommittees for CNS Germ Cell Tumors as well as
Low grade glioma.
Signature applicant / first investigator
AYN Schouten-van Meeteren
Kinderarts oncoloog
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